Production of high activity fibrinolytic agents

ABSTRACT

Fibrinolytic material produced from Aspergillus Oryzal B1273 has blood pressure lowering agents removed therefrom by filtering the material through a cross-linked dextran having a water regain value of substantially 5 grams per gram and particle size 50-270 mesh dry basis, U.S. Standard Sieves.  The filter may be developed with an eluting agent such as hydrochloride acid of pH 3.5 to 5.5 and the fractions of high fibrinolytic activity pooled and dried in vacuo from the frozen state.ALSO:Fibrinolytic material produced from Aspergillus oryz  B1273 has blood pressure lowering agents removed therefrom by filtering the material through a cross-linked dextran having a water regain value of substantially 5 grams per gram and particle size 50-270 mesh dry basis, U.S. standard sieves.  The filter may be developed with an eluting agent such as hydrochloric acid of pH 3.5 to 5.5 and the fractions of high fibrinolytic activity pooled and dried in vacuo from the frozen state.

United States Patent M 3,140,984 PRODUCTION OF HIGH ACTIVITY FIBRINO-LYTIC AGENTS Anthony L. Tosoni, Toronto, Ontario, and David A. J. Ives,Weston, Ontario, Canada, assignors of one-half to The Governors of theUniversity of Toronto and onehalf to Astra Pharmaceutical Products,Inc., Worcester, Mass., a corporation of New York No Drawing. Filed Nov.30, 1961, Ser. No. 156,143 6 Claims. (Cl. 19566) This invention relatesto the production of high activity fibrinolytic agents ofmicrobiological origin substantially free of blood pressure loweringagents and to the fibrinolytic agents so produced.

It is now known that certain micro-organisms are the source of a productwhich has a lytic action on fibrin clots of human origin and that forexample Aspergillus oryzae is a source of such product. The growing ofcertain strains of Aspergillus oryzae by deep culture techniques toprovide an important source of such fibrinolytic product forms thesubject matter of one co-pending application Serial No. 156,142, nowabandoned. Another co-pending application Serial No. 156,144, nowabandoned, relates to the separation of fibrinolytic material ofmicrobiological origin from the medium in which it is produced and tothe purification of such separated fibrinolytic material.

While such fibrinolytic material produced up to the present has beenfound to have the above-referred-to beneficial lytic action on fibrinclots, it has also been found to contain blood pressure lowering agentsand it will be appreciated the value of such material would bematerially enhanced if such blood pressure lowering agents were removed.

It is therefore the object of this invention to enable the production offibrinolytic material substantially free from blood pressure loweringagents. Further, it is the object to enable the removal of such pressurelowering agents to be effected from fibrinolytic material whether ofrelative crude form or of highly purified form.

Another important object is to provide not only a fibrinolytic materialfree from such blood pressure reducing material but a material havinghigh fibrinolytic activity.

Another important object is to enable the removal of such blood pressurelowering agents with a relatively simple procedure so that such enhancedfibrinolytic material may be produced in substantial quantities.

The principal feature of the invention resides in filtering thefibrinolytic material through a cross-linked dextran known in the tradeas Sephadex G50, which has a cross linked dextran having a water regainvalue of 5.7 as defined in the Journal Acta histochemica Bd. 11, 1961,page 306.

According to the invention, the starting fibrinolytic material employedis a relatively crude preparation of a substance produced by growingstrains of the mould Aspergillus oryzae, strain B1273, for example, bythe deep culture techniques as set out in said co-pending applicationSerial No. 156,142. This strain B1273 of Aspergillus oryzae is availablefrom the Quartermaster Research and Development Center, Dept. of Army,Natick, Mass. (see Proc. Soc. Exper. Biol. & Med. 99, 505, 1958 and 102,203, 1959). Alternatively, the starting material may be a more purifiedmaterial such as, for example, the purified fibrinolytic materialsdisclosed in said co-pending application Serial No. 156,144.

According to the invention such fibrinolytic material in either thecrude or more purified form is filtered through a column of cross-linkeddextran described un- 3,140,984 Patented July 14, 1964 Ice der thetrademark Sephadex G50. The filter is then developed with an elutingagent and the eluate collected in fractions and the fractions of highfibrinolytic activity may then be pooled and dried in vacuo from thefrozen state to produce fibrinolytic product which not only has a highpotency but also is substantially free from blood pressure loweringagents.

Preferably, according to the invention, the filtering of the startingfibrinolytic material through the cross-linked dextran, Sephadex G50, iscarried out at low temperatures, for example, at temperatures slightlyabove the freezing point of water to provide increased yields of thedesired blood-pressure-lowering-agent-free fibrinolytic material.

The invention will be more fully understood from the following examples:

Example I 1.0 gram of fibrinolytic material having an activity ofapproximately 5000 units per mg. (as determined by a fibrin plate methodof assay as is well understood in the art) (see proceedings of theSociety for Experimental Biology and Medicine, volume 102, 1959, pages201 to 203) but also having the characteristic of reducing the bloodpressure when administered was dissolved in 15 ml. of water and theacidity adjusted to pH 4.8 with hydrochloric acid. This solution wasfiltered through crosslinked dextran (medium Sephadex G50) in a columnapproximately 2" in diameter and 22" in height and having a temperatureslightly above the freezing point of water, about 3 C. to 6 C. Thefilter was developed with an eluting agent consisting of distilled wateradjusted to pH 4.8 by the careful addition of hydrochloric acid. Theeluate was collected in 15-ml fractions. The fractions with highactivity (fractions 33 to 41) were pooled and dried in vacuo from thefrozen state. The yield was mg. of material having a potency ofapproximately 12,000 units per mg. This product was tested and found tobe substantially completely free of any blood pressure lowering agents.

While in the specific example, the eluting agent was adjusted to pH 4.8,in similar examples the pH varied from about ph 3.5 to about pH 5.5 andsimilar yields obtained.

Example II Although the material obtained from Example I has remarkablyhigh activity in addition to being free from blood pressure loweringagents and is suitable for clinical use, its activity may be furtherincreased as follows according to these examples.

A quantity of the material of Example I having a potency of 12,000 unitsper mg. was dialysed against running tap water yielding a fibrinolyticproduct containing about 18,000 units per mg.

Example III 50 mg. of a relatively cruder fibrinolytic material having apotency of between 3,000 and 4,000 units per mg. were dissolved in 10ml. of 8 molar urea solution and the solution filtered through a columnof cross-linked dextran, Sephadex G50, as in Example I. Water was thenrun through the column and approximately thirty 3 ml. fractionscollected. Four of the fractions (fractions 13-16 inclusive) containingmost of the activity were freeze-dried to give 11 mg. of a producthaving fibrinolytic activity of approximately 8,000 units per mg. Again,the use of the cross-linked dextran, Sephadex G50, was found to not onlymaterially increase the potency of the fibrinolytic material but also toremove the blood pressure lowering agents present in the startingmaterial.

It is to be pointed out that the filtering the material through thecross-linked dextran is best accomplished by using the column in arefrigerator as it appears increased yields are obtained when work iscarried out at low temperature, that is, a temperature slightly abovethe freezing point of water.

The process of using cross-linked dextran, Sephadex G50, in accordancewith the invention is thus shown to be a convenient and useful methodfor preparing a product having high fibrinolytic activity and a productfrom which all blood pressure lowering agents have been substantiallycompletely eliminated. It has been found that for efiicient operation ofthese columns on a repetitive basis such as repeating Examples I andIII, it is useful to wash the columns between successive uses withdilute acid, such for example, as 0.1 N hydrochloric acid wherehydrochloric acid was used as the eluate. Subsequently, this acid may beremoved with the eluant which is used for eluting the fibrinolyticmaterial.

While the above examples are typical illustrations of the invention, itis not intended the invention be limited thereto and variations in suchexamples, as will be apparent to those skilled in the art, may be madewithout departing from the spirit of the invention or scope of theappended claims.

What we claim is:

1. A process for purifying fibrinolytic material produced fromAspergillus oryzae B127 3 and removing blood pressure lowering agentstherefrom consisting in filtering an aqueous solution of saidfibrinolytic material through cross-linked dextran known as SephadexG50, developing the filter with an eluting agent and collecting theeluate which contains the desired resulting fibrinolytic material.

2. A process as claimed in claim 1 in which the solution is filteredthrough the cross-linked dextran in a column.

3. A process as claimed in claim 1 in which the solution is filteredthrough the cross-linked dextran while maintaining the temperaturethereof at about from 3 C. to 6 C.

4. A process as claimed in claim 1 in which the eluting agent consistsof distilled water adjusted to have a pH of about 3.5 to about 5.5.

5. A process as claimed in claim 1 in which said collected eluate isdried in vacuo from the frozen state.

6. A process as claimed in claim 5 in which the product resulting fromsaid drying is dialysed.

References Cited in the file of this patent Proceedings of the Societyfor Experimental Biology and Medicine, volume 99 (1958), pages 504-507;volume 102 (1959), pages 201-203.

Porath et al.: Nature 191, 69-70, July 1961.

Flodin et al.: Nature 188, 493-494, November 5, 1960.

1. A PROCESS FOR PURIFYING FIBRINOLYTIC MATERIAL PRODUCED FROMASPERGILLUS ORYZAE B1273 AND REMOVING BLOOD PRESSURE LOWERING AGENTSTHEREFROM CONSISTING IN FILTERING AN AQUEOUS SOLUTION OF SAIDFIBRINOLYTIC MATERIAL THROUGH CROSS-LINKED DEXTRAN KNOWN AS SEPHADEX G50DEVELOPING THE FILTER WITH AN ELUTING AGENT AND COLLECTING THE ELUATEWHEN CONTAINS THE DESIRED RESULTING FIBRINOLYTIC MATERIAL.